Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (ß-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCß), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in ß-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

Tamm-Horsfall protein and urinary exosome isolation.

Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical.

Large-scale proteomics and phosphoproteomics of urinary exosomes.

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Isolation and purification of exosomes in urine.

Exosomes represent an important and readily isolated subset of the urinary proteome that has the potential to shed much insight on the health status of the kidney. Each segment of the nephron sheds exosomes into the urine. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Two differential centrifugation methods are available for simple purification of exosomes: one uses ultracentrifugation, and the other uses a nanomembrane concentrator. Validation methods include western blots of pan-exosome markers and segment-specific exosome markers, and negative staining electron microscopy.

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct.

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n=22) or immunoblotting (n=6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.

Molecular proximity of cystic fibrosis transmembrane conductance regulator and epithelial sodium channel assessed by fluorescence resonance energy transfer.

We present the evidence for a direct physical association of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), two major ion channels implicated in the pathophysiology of cystic fibrosis, a devastating inherited disease. We employed fluorescence resonance energy transfer, a distance-dependent imaging technique with capability to detect molecular complexes with near angstrom resolution, to estimate the proximity of CFTR and ENaC, an essential variable for possible physical interaction to occur. Fluorescence resonance energy transfer studies were complemented with a classic biochemical approach: coimmunoprecipitation. Our results place CFTR and ENaC within reach of each other, suggestive of a direct interaction between these two proteins.

Prospects for urinary proteomics: exosomes as a source of urinary biomarkers.

Recent progress in biotechnology offers the promise of better medical care at lower costs. Among the techniques that show the greatest promise is mass spectrometry of proteins, which can identify proteins present in body fluids and tissue specimens at a large scale. Because urine can be collected in large amounts in a non-invasive fashion, the potential exists to use mass spectrometry to discover urinary biomarkers that are early predictors of renal disease, or useful in making therapeutic choices. Recently, the authors demonstrated that both membrane proteins and cytosolic proteins from renal epithelia are highly enriched in low-density urinary structures identified as exosomes. Exosomes were found to contain many disease-associated proteins including aquaporin-2, polycystin-1, podocin, non-muscle myosin II, angiotensin-converting enzyme, Na+ K+ 2Cl- cotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC). Potentially, other disease biomarkers could be discovered by mass spectrometry-based proteomic studies in well-defined patient populations. Herein is described the advantages of using urinary exosomes as a starting material for biomarker discovery. In addition, the purpose of this review is to present an overall strategy for biomarker discovery in urine using exosomes and for developing cost-effective clinical assays for these biomarkers, which can potentially be used for early detection of disease, as a means of differential diagnosis, or as a means of guiding therapy. Finally, potential barriers that need to be overcome before urinary proteomics can be applied clinically, are emphasized.